Among all cancers, metastatic melanoma is one of the most deadly. In most cases melanoma develops due to multiple gene mutations induced in skin melanocytes by exposure to UV light, but few of these mutations have been studied in detail. Most melanomas harbor mutations that constitutively activate the RAS-RAF-MEK-ERK pathway, such as the frequent NRAS and BRAF mutations, which are well known to play a critical role in melanoma malignancy. However, mutations in other pathways such as the PTEN, TP53 or CDKN2A tumor suppressors are also needed for melanoma development. Furthermore, recent studies have uncovered new mutations in other gene families that contribute to melanoma malignancy. The objective of the proposed research is to explore the novel hypothesis that Eph receptor mutations play a functional role in melanoma development and/or progression. Recent sequencing studies of ~300 melanoma specimens and cell lines have revealed that approximately half of the tumors carry one or more nonsynonymous mutations in genes of the Eph receptor tyrosine kinase family. The Eph receptors regulate many fundamental biological and pathological processes, such as the growth and invasiveness of cancer cells, and several of them have been implicated in melanoma pathogenesis. Various in silico analyses predict that many of the Eph receptor mutations contribute to malignancy, consistent with our preliminary studies showing that several EphA2 and EphA3 melanoma mutations analyzed so far drastically affect receptor functional properties. We therefore propose two aims to explore the functional significance of Eph mutations in melanoma. In Aim 1, we will characterize a subset of the mutations identified to determine if they affect the abilit of Eph receptors to perform their normal functions, including trafficking to the cell surface, binding ephrin ligands, assembling into receptor signaling clusters, and fulfilling their role as tyrosine kinases. We will especially focus on Eph receptors that are either known or very likely to play a role in melanoma pathogenesis and on the mutations most likely to be driver mutations. In Aim 2, we will investigate how representative Eph mutations affect melanoma cell development and/or malignancy by introducing mutant and wild-type receptors in melanocytes and melanoma cells, and measuring cell proliferation/survival and migration/invasiveness. Surveying the functional effects of Eph receptor mutations occurring in melanoma will provide valuable information on how melanoma cells acquire their malignant properties and on the involvement of the Eph receptor family in cancer development and progression, which is currently incompletely understood. This information, and the collection of research tools to be generated, will also enable future more extensive studies to characterize the importance of normal as well as mutated Eph receptors in additional cell culture and in vivo melanoma models. Cataloguing the effects of Eph gene mutations may also prove useful for identifying novel biomarkers as well as genomic alterations to help prognosis and the design of individualized therapies for melanoma patients.